S1P was purchased from Sigma-Aldrich (St. Louis, MO), and FTY720 was generously provided by Novartis (Basel, Switzerland). All other reagents were obtained from Sigma-Aldrich, unless otherwise noted. Immunofluorescent and Western blotting reagents were obtained as follows: Ivacaftor Texas Red phalloidin (Invitrogen, Carlsbad, CA); and rabbit anti-diphosphorylated MLC, rabbit anti-pan-MLC, rabbit anti-phosphorylated ERK, rabbit anti-pan-ERK (Cell Signaling, Beverly, MA). The labeled dextran vascular permeability assay kit was purchased from Millipore Corporation (Bedford, MA). Fura-2/acetoxymethyl ester was obtained through Invitrogen. Pertussis toxin and genistein were purchased from EMD Biosciences (San Diego, CA). FTY720 Analog Synthesis. Analogs were synthesized as described elsewhere (Lu et al.

, 2009). Cell Culture. Human pulmonary artery endothelial cells (HPAEC) were obtained from Lonza Walkersville, Inc. (Walkersville, MD) and were cultured as described previously (Dudek et al., 2004) in the manufacturer��s recommended endothelial growth medium-2 (EGM-2). Cells were grown at 37��C in a 5% CO2 incubator, and passages 6 to 9 were used for experiments. Media were changed 1 day before experimentation. Transendothelial Monolayer Electrical Resistance. EC were grown to confluence in polycarbonate wells containing evaporated gold microelectrodes, and TER measurements were performed using an electrical cell-substrate impedance sensing system (Applied Biophysics, Troy, NY) as described previously in detail (Garcia et al., 2001).

TER values from each microelectrode were pooled as discrete time points and plotted versus time as the mean �� S.E.M. Vascular Permeability Assay. A transendothelial permeability assay was performed as we described previously (Garcia et al., 1986) using labeled tracer flux across confluent EC grown on confluent polycarbonate filters (Vascular Permeability Assay Kit; Millipore Corporation). In brief, EC grown to confluence on Transwell inserts were exposed to agonist stimulation for 1 h. After stimulation, FITC-labeled dextran was added to the luminal compartment for 2 h, and then FITC-dextran clearance across the filter to the abluminal compartment was measured by relative fluorescence excitation at 485 nm and emission at 530 nm. Immunofluorescence. EC were grown on gelatinized coverslips before exposure to various conditions as described for individual experiments.

EC were then fixed in 3.7% formaldehyde for 10 min, permeabilized with 0.25% Triton X-100 for 5 min, washed in PBS, Cilengitide blocked with 2% bovine serum albumin in Tris-buffered saline with Tween 20 for 1 h, and then incubated for 1 h at room temperature with the primary antibody of interest. After washing, EC were incubated with the appropriate secondary antibody conjugated to immunofluorescent dyes (or Texas Red-conjugated phalloidin for actin staining) for 1 h at room temperature.